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This study examined the changes in mathematics achievement in certain content areas of freshmen before and after Covid-19. For this purpose, in 2019 and 2021, the diagnosis assessment and the mid-term and final exam scores for the first semester of the local engineering college students were converted into 100 points and compared in various aspects. As a result, first, the scores seem to confirm a decline in mathematics achievement in 2021 when compared to the scores achieved before the Covid-19 pandemic. Second, in the case of the upper group, online classes were more effective concerning the study of transcendental function, function and limit, derivatives, and the application of derivatives. Third, in the lower level group, the face-to-face classes were more effective regarding the study of function, transcendental function, function and limit, and integrals. Fourth, the results of the comparison of contents between the upper and lower level groups in 2019 showed that the lower level group’s achievement was higher than the upper level group’s in all areas, but that there was no statistically significant difference. Finally, the results of the comparison between the upper and lower level groups in 2021 showed that the upper level group’s achievement was higher than the lower level group’s in all areas, but that there was a statistically significant difference only when it came to the learning of integrals., in addition to the content characteristics of courses in the liberal arts area regarding the basic nature of a major.

Cytochrome b5 reductase 3 (CYB5R3) is involved in various cellular metabolic processes, including fatty acid synthesis and drug metabolism. However, the role of CYB5R3 in cancer development remains poorly understood. Here, we show that CYB5R3 expression is downregulated in human lung cancer cell lines and tissues. Adenoviral overexpression of CYB5R3 suppresses lung cancer cell growth in vitro and in vivo. However, CYB5R3 deficiency promotes tumorigenesis and metastasis in mouse models. Transcriptome analysis revealed that apoptosis- and endoplasmic reticulum (ER) stress-related genes are upregulated in CYB5R3-overexpressing lung cancer cells. Metabolomics analysis revealed that CYB5R3 overexpression increased the production of NAD+ and oxidized glutathione (GSSG). Ectopic CYB5R3 is mainly localized in the ER, where CYB5R3-dependent ER stress is induced by activating protein kinase RNA-like ER kinase (PERK) and inositol-requiring enzyme 1 alpha (IRE1α). Moreover, NAD+ activates poly (ADP-ribose) polymerase16 (PARP16), an ER-resident protein, to promote ADP-ribosylation of PERK and IRE1α and induce ER stress. In addition, CYB5R3 induces the generation of reactive oxygen species and caspase-9-dependent-intrinsic cell death. Our findings highlight the significance of CYB5R3 as a tumor suppressor for the development of CYB5R3-based therapeutics for lung cancer.

Neural stem/progenitor cells (NSPCs) contribute to the physiological cellular turnover of the adult brain and make up its regenerative potential. It is thus essential to understand how different factors influence their proliferation and differentiation to gain better insight into potential therapeutic targets in neurodegenerative diseases and traumatic brain injuries. Recent evidences indicate the roles of redox stress sensing and coping mechanisms in mediating the balance between NSPC self-renewal and differentiation. Such mechanisms involve direct cysteine modification, signaling and metabolic reprogramming, epigenetic alterations and transcription changes leading to adaptive responses like autophagy. Here, we discuss emerging findings on the involvement of redox sensors and effectors and their mechanisms in influencing changes in cellular redox potential and NSPC fate.

EGFR is frequently amplified, mutated, and overexpressed in malignant gliomas. Yet the EGFR-targeted therapies have thus far produced only marginal clinical responses, and the underlying mechanism remains poorly understood. Using an inducible oncogenic EGFR-driven glioma mouse model system, our current study reveals that a small population of glioma cells can evade therapy-initiated apoptosis and potentiate relapse development by adopting a mesenchymal-like phenotypic state that no longer depends on oncogenic EGFR signaling. Transcriptome analyses of proximal and distal treatment responses identified TGFβ/YAP/Slug signaling cascade activation as a major regulatory mechanism that promotes therapy-induced glioma mesenchymal lineage transdifferentiation. Following anti-EGFR treatment, TGFβ secreted from stressed glioma cells acted to promote YAP nuclear translocation that stimulated upregulation of the pro-mesenchymal transcriptional factor SLUG and subsequent glioma lineage transdifferentiation toward a stable therapy-refractory state. Blockade of this adaptive response through suppression of TGFβ-mediated YAP activation significantly delayed anti-EGFR relapse and prolonged animal survival. Together, our findings shed new insight into EGFR-targeted therapy resistance and suggest that combinatorial therapies of targeting both EGFR and mechanisms underlying glioma lineage transdifferentiation could ultimately lead to deeper and more durable responses. Significance: This study demonstrates that molecular reprogramming and lineage transdifferentiation underlie anti-EGFR therapy resistance and are clinically relevant to the development of new combinatorial targeting strategies against malignant gliomas with aberrant EGFR signaling.

Despite recent therapeutic advances, prostate cancer remains a leading cause of cancer-related death. A subset of castration-resistant prostate cancers become androgen receptor (AR) signaling independent and develop neuroendocrine prostate cancer (NEPC) features through lineage plasticity. These NEPC tumors, associated with aggressive disease and poor prognosis, are driven, in part, by aberrant expression of N-Myc, through mechanisms that remain unclear. Integrative analysis of the N-Myc transcriptome, cistrome, and interactome using in vivo, in vitro, and ex vivo models (including patient-derived organoids) identified a lineage switch towards a neural identity associated with epigenetic reprogramming. N-Myc and known AR cofactors (e.g., FOXA1 and HOXB13) overlapped, independently of AR, at genomic loci implicated in neural lineage specification. Moreover, histone marks specifically associated with lineage-defining genes were reprogrammed by N-Myc. We also demonstrated that the N-Myc–induced molecular program accurately classifies our cohort of patients with advanced prostate cancer. Finally, we revealed the potential for enhancer of zeste homolog 2 (EZH2) inhibition to reverse the N-Myc–induced suppression of epithelial lineage genes. Altogether, our data provide insights into how N-Myc regulates lineage plasticity and epigenetic reprogramming associated with lineage specification. The N-Myc signature we defined could also help predict the evolution of prostate cancer and thus better guide the choice of future therapeutic strategies.

Chronic kidney disease (CKD) has become epidemic worldwide. Mitochondrial reactive oxygen species (ROS)-induced oxidative stress is an important mediator of CKD, and Prx3 plays a critical role in maintenance of mitochondrial ROS. The present study examined the role of Prx3 in the context of fibrosis, a common feature of CKD, using Prx3 KO mice under obstructive and diabetic stress. Prx3 deficiency accelerated fibrosis and inflammation accompanied by mitochondrial oxidative stress in obstructed and diabetic kidneys as well as in proximal tubular epithelial (mProx) cells. In addition, Prx3 deficiency induced Raw264.7 macrophages activation, leading to upregulation of proinflammatory cytokines. Conditioned media from LPS-stimulated Prx3 deficient macrophages accelerated proinflammatory and profibrotic cytokines in mProx cells. Interestingly, Prx3 deficiency induced most inflammatory and fibrotic cytokines at basal condition in both tissues and cells. Taken together, these results demonstrate that Prx3 deficiency can accelerate CKD through interactions between macrophages and tubular epithelial cells.

Neural stem/progenitor cells (NSPCs) persist over the lifespan while encountering constant challenges from age or injury related brain environmental changes like elevated oxidative stress. But how oxidative stress regulates NSPC and its neurogenic differentiation is less clear. Here we report that acutely elevated cellular oxidative stress in NSPCs modulates neurogenic differentiation through induction of Forkhead box protein O3 (FOXO3)-mediated cGAS/STING and type I interferon (IFN-I) responses. We show that oxidative stress activates FOXO3 and its transcriptional target glycine-N-methyltransferase (GNMT) whose upregulation triggers depletion of s-adenosylmethionine (SAM), a key co-substrate involved in methyl group transfer reactions. Mechanistically, we demonstrate that reduced intracellular SAM availability disrupts carboxymethylation and maturation of nuclear lamin, which induce cytosolic release of chromatin fragments and subsequent activation of the cGAS/STING-IFN-I cascade to suppress neurogenic differentiation. Together, our findings suggest the FOXO3-GNMT/SAM-lamin-cGAS/STING-IFN-I signaling cascade as a critical stress response program that regulates long-term regenerative potential., Neural stem and progenitor cells (NSPCs) encounter constant stresses during aging, such as elevated oxidative stress. Here the authors show that oxidative stress induced reduction in NSPC neural differentiation is mediated by a FOXO3-GNMT/SAM-lamin-cGAS/STING-IFN-I signalling cascade initiated by FOXO3 oxidation.

Epidermal growth factor receptor (EGFR) is frequently amplified, mutated and overexpressed in malignant gliomas. Yet the EGFR-targeted therapies have thus far produced only marginal clinical response, and the underlying mechanism remains poorly understood. Through analyses of an inducible oncogenic EGFR-driven glioma mouse model system, our current study reveals a small population of glioma cells that can evade therapy-initiated apoptosis and potentiate relapse development by adopting a mesenchymal-like phenotypic state that no longer depends on oncogenic EGFR signaling. Transcriptome analyses of proximal and distal treatment responses further identify TGFβ/YAP/Slug signaling cascade activation as major regulatory mechanism that promotes therapy-induced glioma mesenchymal lineage transdifferentiation. Following anti-EGFR treatment, the TGFβ secreted from the stressed glioma cells acts to promote YAP nuclear translocation and activation, which subsequently stimulates upregulation of the pro-mesenchymal transcriptional factor Slug and then glioma lineage transdifferentiation towards a stable therapy-refractory state. Blockade of this adaptive response through enforced dominant negative YAP expression significantly delayed anti-EGFR relapse and significantly prolonged animal survival. Together, our findings shed new insight into EGFR-targeted therapy resistance and suggest that combinatorial therapies of targeting both EGFR and mechanisms underlying glioma lineage transdifferentiation could ultimately lead to deeper and more durable responses.SignificanceThis study demonstrates that molecular reprogramming and lineage transdifferentiation underlie anti-EGFR therapy resistance and is clinically relevant to the development of new combinatorial targeting strategies against malignant gliomas carrying aberrant EGFR signaling.

Capicua (CIC), a member of the high mobility group-box (HMG-box) superfamily of transcriptional repressors, is frequently mutated in human oligodendrogliomas. However, its functions in brain development and tumorigenesis remain poorly understood. Here, we report that brain-specific deletion of Cic compromises developmental transition of neuroblasts to immature neurons in mouse hippocampus and compromises normal neuronal differentiation. Combined gene expression and ChIP-seq analyses identified VGF as an important CIC-repressed transcriptional surrogate involved in neuronal lineage regulation. Aberrant VGF expression promotes neural progenitor cell proliferation by suppressing their differentiation. Mechanistically, we demonstrated that CIC represses VGF expression by tethering SIN3-HDAC to form a transcriptional corepressor complex. Mass spectrometry analysis of CIC-interacting proteins further identified the BRG1-containing mSWI/SNF complex whose function is necessary for transcriptional repression by CIC. Together, this study uncovers a potentially novel regulatory pathway of CIC-dependent neuronal differentiation and may implicate these molecular mechanisms in CIC-dependent brain tumorigenesis.

Neural stem/progenitor cells (NSPCs) persist over the lifespan while encountering constant challenges from age or injury related brain environmental changes, including elevated oxidative stress. A time-dependent stress response that regulates the dynamic balance between quiescence and differentiation is thus essential to preserve NSPC long-term regenerative potential. Here we report that acutely elevated cellular oxidative stress in NSPCs suppresses neurogenic differentiation through induction of FOXO3-mediated cGAS/STING and type I interferon (IFN-I) responses. We show that oxidative stress activates FOXO3 promoting upregulation of its transcriptional target glycine-N-methyltransferase (GNMT) and thus depletion of s-adenosylmethionine (SAM), a key co-substrate involved in methyl group transfer reactions. Mechanistically, we demonstrate that reduced intracellular SAM availability disrupts carboxymethylation and maturation of nuclear lamin, which trigger cytosolic release of chromatin fragments and subsequent activation of the cGAS/STING/IFN-I cascade. Together, our findings suggest the FOXO3-GNMT/SAM-lamin-cGAS/STING-IFN-I signaling cascade as a critical stress response program that preserves its long-term regenerative potential.

Summary Loss of a cell's ability to terminally differentiate because of mutations is a selected genetic event in tumorigenesis. Genomic analyses of low-grade glioma have reported recurrent mutations of far upstream element-binding protein 1 (FUBP1). Here, we show that FUBP1 expression is dynamically regulated during neurogenesis and that its downregulation in neural progenitors impairs terminal differentiation and promotes tumorigenesis collaboratively with expression of IDH1R132H. Mechanistically, collaborative action between SRRM4 and FUBP1 is necessary for mini-exon splicing of the neurospecific LSD1+8a isoform. LSD1+8a was downregulated upon loss of FUBP1 in neural progenitors, thereby impairing terminal neuronal differentiation and maturation. Reinforcing LSD1+8a expression in FUBP1-downregulated neural progenitors restored terminal differentiation and suppressed tumorigenesis; hence, LSD1+8a is an obligatory effector of FUBP1-dependent neuronal differentiation. These findings establish a direct role for FUBP1 in neuronal differentiation and also explain its tumor-suppressor function in the nervous system., Graphical Abstract, Highlights • FUBP1 is the tumor suppressor necessary for terminal differentiation of neurons • FUBP1 promotes inclusion of neuron-specific LSD1 exon 8a • FUBP1 and IDH1 mutations converge on impaired differentiation of neural progenitors, In this article, Paik and colleagues demonstrate that FUBP1 plays an indispensable role in promoting terminal differentiation of neurons and that lack of FUBP1 interferes with early-born neuronal cells exiting the cell cycle and predisposes these cells for transformation. These findings explain how FUBP1 serves uniquely as a tumor suppressor in the CNS.

Mantle cell lymphoma (MCL) often has an adverse prognosis and despite aggressive multimodal treatment with conventional and targeted therapies, the median survival of MCL patients remains approximately 4 years. Thus, there is a significant unmet need to find novel targets and rational combination treatments. Targeting lineage vulnerabilities driven by specific transcription factors has been broadly confirmed as an effective intervention in many human cancers. To identify the transcription program dependency of MCL cells, we conducted an unbiased domain-focused CRISPR-Cas9 screening against a library of 8,750 sgRNAs targeting 1,434 transcription factors in MCL cell lines (JEKO1, MAVER1, UPN1, and CCMCL1). We identified Forkhead Box O1 (FOXO1), EBF1, PAX5, and IRF4 as 4 transcription factors that are specifically required for MCL survival and growth. Chromatin-immunoprecipitation and sequencing (ChIP-Seq) analysis further revealed that the four transcription factors act together to orchestrate B cell lineage transcriptional program and MCL cell survival. Despite its well-recognized role as a tumor suppressor, FOXO1 has been implicated as a lineage specific transcription factor involved in mature B cell development. Genetic studies revealed a critical role of FOXO1 in germinal center dark zone formation and lymphomagenesis, raising the possibility that FOXO1 acts as a master transcription factor for lineage survival transcription program of MCL. Indeed, hierarchical interaction analysis revealed that FOXO1 functions as a pioneer factor that facilitate the chromatin access of other B cell lineage transcription factors. We demonstrated that interaction of FOXO1 to its cognate motif stabilizes B cell transcription factor complex and supports MCL progression. Along this line, we show that enforced expression of FOXO1 in myeloid leukemia cells induces transdifferentiation and B-cell specific gene expression. Mechanistically, we demonstrate through tiling CRISPR scanning screen that forkhead DNA binding and c-terminal transactivation domains of FOXO1 are specifically required for the viability of MCL cells. Given our finding of FOXO1 as a lineage-specific oncogene in MCL, we next explored the possibility of developing FOXO1-tageted inhibitors. We screened a library of potential small molecule inhibitors of FOXO1 (Forkhead BioTherapeutics) and identified cpd10 as one of the most potent and selective FOXO1 inhibitors (IC 50=76 ×/÷ 1.7. nM) Through the CCMCL1 MCL-NSG preclinical model, we found that cpd10 (100 mg/kg/daily) was well tolerated without overt toxicities. Importantly, prolonged treatment induced a robust cytotoxic response of MCL cells and suppressed MCL progression in vivo. Altogether, our findings identified FOXO1 as a MCL lineage-survival oncogene that can be exploited as a therapeutic target of future drug development. Disclosures Cantley: Petra Pharmaceuticals: Research Funding; Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Elemento: Freenome: Consultancy, Other: Current equity holder in a privately-held company; Volastra Therapeutics: Consultancy, Other: Current equity holder, Research Funding; Champions Oncology: Consultancy; Janssen: Research Funding; Owkin: Consultancy, Other: Current equity holder; AstraZeneca: Research Funding; One Three Biotech: Consultancy, Other: Current equity holder; Eli Lilly: Research Funding; Johnson and Johnson: Research Funding. Baiocchi: Prelude Therapeutics: Consultancy; viracta: Consultancy, Current holder of stock options in a privately-held company; Codiak Biosciences: Research Funding; Atara Biotherapeutics: Consultancy. Belvedere: Forkhead BioTherapeutics: Current Employment. Paik: Forkhead BioTherapeutics: Research Funding.

Mantle cell lymphoma (MCL) is an incurable B cell malignancy, comprising 5% of non-Hodgkin lymphomas diagnosed annually. MCL is associated with a poor prognosis due to emergence of resistance to immuno-chemotherapy and targeted agents. The average overall survival of patients with MCL is 4-6 years and for the majority of patients who progress on targeted agents, survival remains at a dismal 3-8 months. There is a major unmet need to identify new therapeutic approaches that are well tolerated to improve treatment outcomes and quality of life. The type II protein arginine methyltransferase enzyme, PRMT5 is overexpressed and promotes growth and survival of MCL. Inhibition of PRMT5 with a novel, SAM-competitive class of inhibitors drives anti-tumor activity in MCL cell lines and patient derived xenograft (PDX) models derived from patients with relapse or refractory disease. Selective inhibition of PRMT5 with PRT-382 (Prelude Therapeutics) in these models and MCL cell lines leads to disruption of constitutive PI3K/AKT signaling, dephosphorylation and nuclear translocation of FOXO1, and enhanced recruitment of this tumor suppressor protein to target genes. By performing chromatin immunoprecipitation-sequencing (ChIP seq) analysis, we identified over 800 newly emerged FOXO1-bound genomic loci, including multiple pro-apoptotic BCL2 family proteins (BAX, BAK1, BIK, BBC3, BMF and NOXA1). FOXO1 localization and transcriptional differences were confirmed by ChIP PCR and RT-PCR respectively. Protein levels were measured with Western blotting. BAX was identified as the most common direct target of FOXO1-transriptional activity that was upregulated on both a transcript and protein level. This led us to hypothesize that PRMT5 inhibition could potentially drive a therapeutic vulnerability to the BCL-2 inhibitor venetoclax. Single agent and combination treatment with venetoclax and PRT382 was performed in nine MCL lines. Of the nine lines, four were considered relatively resistant to PRT-382 and five resistant to venetoclax. Synergy scores, determined from MTS assays, showed significant levels of synergy in the majority of MCL lines tested. CCMCL1 and UPN1, BCL-2 negative MCL lines, and Maver1, which is highly resistant to PRMT5i, were the only cell lines to not show synergy. The cell line with the highest levels of synergy, Z-138, expressed high levels of BCL-2 and is ibrutinib resistant. Overall, there was a strong positive correlation between BCL-2 expression and synergy score (r= -0.8956, p=0.0064). The synergy seen was confirmed to be through the intrinsic apoptotic pathway based on caspase activity. To determine a mechanism of action, BAX and BAK1 were knocked down in four cell lines, three that displayed synergy and one that was resistant. This suggests that BAX expression is essential for synergy between PRMT5 and BCL2 inhibition to occur. Knock down of BAK1, the other effector of the BCL2 family of proteins, did not show protection suggesting that BAX is necessary and sufficient for this therapeutic synergy to occur. We also determined that p53 status did not correlate to the response seen (p=0.477), supporting that this mechanism is occurring through FOXO1 transcriptional regulation. In vivo evaluation in two preclinical MCL models showed therapeutic synergy with combination venetoclax/PRT382 treatment. Mice were treated with sub-therapeutic doses of venetoclax and/or PRT382 and disease burden was assessed weekly via flow cytometry. Combination treatment with well-tolerated doses of venetoclax and PRMT5 inhibitors in the MCL in vivo models showed synergistic anti-tumor activity. Both PDX models showed an extension of life with combination treatment (P Disclosures Zhang: Prelude Therapeutics: Current Employment. Vaddi: Prelude Therapeutics: Current Employment, Current equity holder in publicly-traded company. Elemento: AstraZeneca: Research Funding; Janssen: Research Funding; Johnson and Johnson: Research Funding; One Three Biotech: Consultancy, Other: Current equity holder; Volastra Therapeutics: Consultancy, Other: Current equity holder, Research Funding; Eli Lilly: Research Funding; Freenome: Consultancy, Other: Current equity holder in a privately-held company; Owkin: Consultancy, Other: Current equity holder; Champions Oncology: Consultancy. Scherle: Prelude Therapeutics: Current Employment, Current equity holder in publicly-traded company. Paik: Forkhead BioTherapeutics: Research Funding. Baiocchi: Prelude Therapeutics: Consultancy; viracta: Consultancy, Current holder of stock options in a privately-held company; Codiak Biosciences: Research Funding; Atara Biotherapeutics: Consultancy.

Capicua (CIC), a member of the high mobility group (HMG)-box superfamily of transcriptional repressors, is frequently mutated in human oligodendrogliomas. But its function in brain development and tumorigenesis remains poorly understood. Here, we report that brain-specific deletion of Cic compromises developmental transition of neuroblast to immature neurons in mouse hippocampus and compromises normal neuronal differentiation. Combined gene expression and ChIP-seq analyses identified VGF as an important CIC-repressed transcriptional surrogates involved in neuronal lineage regulation. Aberrant VGF expression promotes neural progenitor cell proliferation by suppressing their differentiation. Mechanistically, we demonstrated that CIC represses VGF expression by tethering SIN3-HDAC to form a transcriptional corepressor complex. Mass spectrometry analysis of CIC-interacting proteins further identified BRG1 containing mSWI/SNF complex of which function is necessary for transcriptional repression by CIC. Together, this study uncovers a novel regulatory pathway of CIC-dependent neuronal differentiation and provides molecular insights into the etiology of CIC-dependent brain tumors.

Peroxisomes are essential organelles for maintaining the homeostasis of lipids and reactive oxygen species (ROS). While oxidative stress-induced endoplasmic reticulum (ER) stress plays an important role in nonalcoholic fatty liver disease (NAFLD), the role of peroxisomes in ROS-mediated ER stress in the development of NAFLD remains elusive. We investigated whether an impaired peroxisomal redox state accelerates NAFLD by activating ER stress by inhibiting catalase, an antioxidant expressed exclusively in peroxisomes. Wild-type (WT) and catalase knockout (CKO) mice were fed either a normal diet or a high-fat diet (HFD) for 11 weeks. HFD-induced phenotype changes and liver injury accompanied by ER stress and peroxisomal dysfunction were accelerated in CKO mice compared to WT mice. Interestingly, these changes were also significantly increased in CKO mice fed a normal diet. Inhibition of catalase by 3-aminotriazole in hepatocytes resulted in the following effects: (i) increased peroxisomal H2O2 levels as measured by a peroxisome-targeted H2O2 probe (HyPer-P); (ii) elevated intracellular ROS; (iii) decreased peroxisomal biogenesis; (iv) activated ER stress; (v) induced lipogenic genes and neutral lipid accumulation; and (vi) suppressed insulin signaling cascade associated with JNK activation. N-acetylcysteine or 4-phenylbutyric acid effectively prevented those alterations. These results suggest that a redox imbalance in peroxisomes perturbs cellular metabolism through the activation of ER stress in the liver.

Epidermal growth factor receptor (EGFR) is frequently amplified, mutated and overexpressed in malignant gliomas. Our investigation of the proximal and distal responses to EGFR inhibition identified the molecular mechanisms for the therapeutic resistance of EGFR inhibition in gliomas. Oncogenic EGFRviii-dependent gliomas initially showed cytotoxic responses upon removal of oncogenic cue. However, during the initial regression, a subset of tumor cells with activated mesenchymal subtype gene expression program emerged by lineage reprogramming which promoted tumor relapse in the absence of EGFRviii signaling. This lineage switch is dependent on YAP1 activation as a response to therapy, and is a key to EGFRviii independent tumor growth. YAP signature stratifies overall survival of recurrent glioma patients. Inhibition of YAP1 activation suppressed mesenchymal gene expression and significantly delayed recurrence of gliomas. Our findings provide mechanisms underlying inefficacies of EGFR targeted therapy in glioblastoma and suggest a new combinatorial targeting of EGFR and YAP for deeper and more durable responses.

Summary Neurodegeneration resulting in cognitive and motor impairment is an inevitable consequence of aging. Little is known about the genetic regulation of this process despite its overriding importance in normal aging. Here, we identify the Forkhead Box O (FOXO) transcription factor 1, 3, and 4 isoforms as a guardian of neuronal integrity by inhibiting age‐progressive axonal degeneration in mammals. FOXO expression progressively increased in aging human and mouse brains. The nervous system‐specific deletion of Foxo transcription factors in mice accelerates aging‐related axonal tract degeneration, which is followed by motor dysfunction. This accelerated neurodegeneration is accompanied by levels of white matter astrogliosis and microgliosis in middle‐aged Foxo knockout mice that are typically only observed in very old wild‐type mice and other aged mammals, including humans. Mechanistically, axonal degeneration in nerve‐specific Foxo knockout mice is associated with elevated mTORC1 activity and accompanying proteotoxic stress due to decreased Sestrin3 expression. Inhibition of mTORC1 by rapamycin treatment mimics FOXO action and prevented axonal degeneration in Foxo knockout mice with accelerated nervous system aging. Defining this central role for FOXO in neuroprotection during mammalian aging offers an invaluable window into the aging process itself.

Loss of terminal differentiation capacity through genetic mutations is a selected genetic event in tumorigenesis. 1p19q co-deletion associated recurrent mutations of far upstream element-binding protein 1 (FUBP1) have been reported from genomic analysis of low- grade oligodendroglioma. It is frequently overexpressed in many human cancers and strongly correlated with disease progression. However, left unanswered is how FUBP1 serves uniquely as a tumor suppressor in the central nervous system. Here we show that expression of FUBP1 is dynamically regulated during neurogenesis, marking specific stages of neuronal differentiation. Loss of FUBP1 in neural stem/progenitor cells (NPC) increased the number of cells fail to terminally differentiate. Knockdown of FUBP1 further blocks differentiation and promotes tumorigenesis collaboratively with the expression of neomorphic isocitrate dehydrorgenase 1 mutant IDH1R132H. Mechanistically FUBP1 is necessary for mini exon 8a splicing of neurospecific isoform of histone demethylase LSD1 (nLSD1). It binds to intron 8 of LSD1 together with a neuron-specific splicing complex containing NOVA1 and 2 and works synergistically with SRRM4 for the inclusion of mini exon 8a. The expression level of nLSD1 is decreased upon loss of FUBP1 in neural progenitors, impairing the terminal neuronal differentiation and maturation. Reinforcing nLSD1 expression in FUBP1-downregulated NPC restores terminal differentiation, establishing nLSD1 as an obligatory effector of FUBP1-dependent neuronal differentiation. Together, these findings establish a direct role for FUBP1 in neuronal differentiation and also uncover the mechanism for its tumor suppressor function in the nervous system.

Forkhead transcription factor family O (FoxO) maintains adult stem cell reserves by supporting their long-term proliferative potential. MicroRNAs (miRs) regulate neuronal stem/progenitor cell (NSPC) proliferation and differentiation during neural development by controlling the expression of a specific set of target genes. In the neurogenic subventricular zone, FoxO1 is specifically expressed in NSPCs and is no longer detected during the transition to neuroblast stage, forming an inverse correlation with miR-9 expression. The 3'-untranslated region of FoxO1 contains a conserved target sequence of miR-9 and FoxO1 expression is coordinated in concert with miR-9 during neuronal differentiation. Our study demonstrates that FoxO1 contributes to NSPC fate decision through its cooperation with the Notch signaling pathway.

Loss of the histone H3.3-specific chaperone component ATRX or its partner DAXX frequently occurs in human cancers that employ alternative lengthening of telomeres (ALT) for chromosomal end protection, yet the underlying mechanism remains unclear. Here, we report that ATRX/DAXX does not serve as an immediate repressive switch for ALT. Instead, ATRX or DAXX depletion gradually induces telomere DNA replication dysfunction that activates not only homology-directed DNA repair responses but also cell cycle checkpoint control. Mechanistically, we demonstrate that this process is contingent on ATRX/DAXX histone chaperone function, independently of telomere length. Combined ATAC-seq and telomere chromatin immunoprecipitation studies reveal that ATRX loss provokes progressive telomere decondensation that culminates in the inception of persistent telomere replication dysfunction. We further show that endogenous telomerase activity cannot overcome telomere dysfunction induced by ATRX loss, leaving telomere repair-based ALT as the only viable mechanism for telomere maintenance during immortalization. Together, these findings implicate ALT activation as an adaptive response to ATRX/DAXX loss-induced telomere replication dysfunction.

Truncated N(6)-substituted-(N)-methanocarba-adenosine derivatives with 2-hexynyl substitution were synthesized to examine parallels with corresponding 4'-thioadenosines. Hydrophobic N(6) and/or C2 substituents were tolerated in A3AR binding, but only an unsubstituted 6-amino group with a C2-hexynyl group promoted high hA2AAR affinity. A small hydrophobic alkyl (4b and 4c) or N(6)-cycloalkyl group (4d) showed excellent binding affinity at the hA3AR and was better than an unsubstituted free amino group (4a). A3AR affinities of 3-halobenzylamine derivatives 4f-4i did not differ significantly, with Ki values of 7.8-16.0 nM. N(6)-Methyl derivative 4b (Ki = 4.9 nM) was a highly selective, low efficacy partial A3AR agonist. All compounds were screened for renoprotective effects in human TGF-β1-stimulated mProx tubular cells, a kidney fibrosis model. Most compounds strongly inhibited TGF-β1-induced collagen I upregulation, and their A3AR binding affinities were proportional to antifibrotic effects; 4b was most potent (IC50 = 0.83 μM), indicating its potential as a good therapeutic candidate for treating renal fibrosis.

Background: Despite recent advances in the development of highly effective androgen receptor (AR)-directed therapies for the treatment of prostate cancer, nearly 37% of patients develop resistance. A further third of these patients progress to develop aggressive neuroendocrine prostate cancer (NEPC) for which no effective therapies exist. Lineage plasticity, a process by which differentiated cells lose their identity and acquire alternative lineage programs, has been proposed as a mechanism of resistance to targeted therapies in epithelial tumors such as prostate cancer. Although epigenetic deregulation has been implicated as a driver of the epithelial to neuronal switch observed in NEPC, the molecular programs are poorly understood. Previously, we observed that the majority of NEPC and 20% of castration-resistant prostate cancer (CRPC) aberrantly overexpress the transcription factor MYCN (N-Myc). Despite this frequent occurrence, the role of N-Myc in driving lineage plasticity and the epigenetic mechanisms which regulate disease progression remain to be elucidated. Methods: We analyzed overall survival and whole transcriptome data from a cohort of over 200 prostate cancer patients, including the largest-to-date population of NEPC patients. We also analyzed epigenetic modifications along with the N-Myc transcriptome, cistrome and chromatin-bound interactome by performing ChIP-seq, RNA-seq and RIME in a combination of mouse models, human prostate cancer cell lines, and NEPC patient-derived organoids. Results: Expression of MYCN in CRPC and NEPC patients correlates with reduced overall survival. NEPC tumors are significantly enriched for stem cell genes associated with normal neuroendocrine cell precursors, embryonic stem cells and neural lineage-defining genes from activated neural stem cells. The integration of next-generation sequencing data revealed that the N-Myc cistrome is androgen-dependent and drives a transcriptional program leading to epithelial plasticity and the acquisition of clinically relevant neuronal lineage markers. Our data also reveal that N-Myc interacting proteins as well as AR co-factors and pioneering factors (e.g. FOXA1 and HOXB13) explain the observed dynamic binding of N-Myc. Interestingly, histone marks and EZH2 activity specifically associated with lineage-defining genes were reprogrammed by N-Myc. Finally, we demonstrated that N-Myc-induced gene expression and epigenetic changes accurately classify our patient cohort. Conclusions: We describe a novel role for N-Myc in prostate cancer, characterized by changes in the N-Myc cistrome and cooperation with AR co-factors, as well as reprogramming of the epigenome in an androgen context-dependent manner. This reprogramming is associated with induction of a lineage plastic state and a switch towards a neural identity that favors the development of AR independence and NEPC. Citation Format: Adeline Berger, Nicholas Brady, Rohan Bareja, Brian Robinson, Vincenza Conteduca, Michael Augello, Loredana Puca, Adnan Ahmed, Xiaodong Lu, Inah Hwang, Alyssa Bagadion, Andrea Sboner, Olivier Elemento, Jihye Paik, Jindan Yu, Christopher Barbieri, Noah Dephoure, Himisha Beltran, David S. Rickman. N-Myc-mediated epigenetic reprogramming drives lineage plasticity in advanced prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2099.

Macrophages are important components of adipose tissue inflammation, which results in metabolic diseases such as insulin resistance. Notably, obesity induces a proinflammatory phenotypic switch in adipose tissue macrophages, and oxidative stress facilitates this switch. Thus, we examined the role of endogenous catalase, a key regulator of oxidative stress, in the activity of adipose tissue macrophages in obese mice. Catalase knockout (CKO) exacerbated insulin resistance, amplified oxidative stress, and accelerated macrophage infiltration into epididymal white adipose tissue in mice on normal or high-fat diet. Interestingly, catalase deficiency also enhanced classical macrophage activation (M1) and inflammation but suppressed alternative activation (M2) regardless of diet. Similarly, pharmacological inhibition of catalase activity using 3-aminotriazole induced the same phenotypic switch and inflammatory response in RAW264.7 macrophages. Finally, the same phenotypic switch and inflammatory responses were observed in primary bone marrow-derived macrophages from CKO mice. Taken together, the data indicate that endogenous catalase regulates the polarization of adipose tissue macrophages and thereby inhibits inflammation and insulin resistance.

Mitochondrial reactive oxygen species (ROS) play an important role in diabetes complications, including diabetic nephropathy (DN). Plasma free fatty acids (FFAs) as well as glucose are increased in diabetes, and peroxisomes and mitochondria participate in FFA oxidation in an interconnected fashion. Therefore, we investigated whether deficiency of catalase, a major peroxisomal antioxidant, accelerates DN through peroxisomal dysfunction and abnormal renal FFA metabolism. Diabetes was induced by multiple injections of low-dose streptozotocin into catalase knock-out (CKO) and wild-type (WT) C57BL/6 mice. Murine mesangial cells (MMCs) transfected with catalase small interfering RNA followed by catalase overexpression were used to further elucidate the role of endogenous catalase. Despite equivalent hyperglycemia, parameters of DN, along with markers of oxidative stress, were more accelerated in diabetic CKO mice than in diabetic WT mice up to 10 weeks of diabetes. CKO mice and MMCs showed impaired peroxisomal/mitochondrial biogenesis and FFA oxidation. Catalase deficiency increased mitochondrial ROS and fibronectin expression in response to FFAs, which were effectively restored by catalase overexpression or N-acetylcysteine. These data provide unprecedented evidence that FFA-induced peroxisomal dysfunction exacerbates DN and that endogenous catalase plays an important role in protecting the kidney from diabetic stress through maintaining peroxisomal and mitochondrial fitness.

Fenofibrate activates not only peroxisome proliferator-activated receptor-α (PPARα) but also adenosine monophosphate-activated protein kinase (AMPK). AMPK-mediated cellular responses protect kidney from high-fat diet (HFD)-induced injury, and autophagy resulting from AMPK activation has been regarded as a stress-response mechanism. Thus the present study examined the role of AMPK and autophagy in the renotherapeutic effects of fenofibrate. C57BL/6J mice were divided into three groups: normal diet (ND), HFD, and HFD + fenofibrate (HFD + FF). Fenofibrate was administered 4 wk after the initiation of the HFD when renal injury was initiated. Mouse proximal tubule cells (mProx24) were used to clarify the role of AMPK. Feeding mice with HFD for 12 wk induced insulin resistance and kidney injury such as albuminuria, glomerulosclerosis, tubular injury, and inflammation, which were effectively inhibited by fenofibrate. In addition, fenofibrate treatment resulted in the activation of renal AMPK, upregulation of fatty acid oxidation (FAO) enzymes and antioxidants, and induction of autophagy in the HFD mice. In mProx24 cells, fenofibrate activated AMPK in a concentration-dependent manner, upregulated FAO enzymes and antioxidants, and induced autophagy, all of which were inhibited by treatment of compound C, an AMPK inhibitor. Fenofibrate-induced autophagy was also significantly blocked by AMPKα1 siRNA but not by PPARα siRNA. Collectively, these results demonstrate that delayed treatment with fenofibrate has a therapeutic effect on HFD-induced kidney injury, at least in part, through the activation of AMPK and induction of subsequent downstream effectors: autophagy, FAO enzymes, and antioxidants.

Loss of terminal differentiation capacity through genetic mutations is a selected genetic event in tumorigenesis. 1p19q co-deletion associated recurrent mutations of far upstream element-binding protein 1 (FUBP1) have been reported from genomic analysis of low- grade oligodendroglioma. It is frequently overexpressed in many human cancers and strongly correlated with disease progression. However, left unanswered is how FUBP1 serves uniquely as a tumor suppressor in the central nervous system. Here we show that expression of FUBP1 is dynamically regulated during neurogenesis, marking specific stages of neuronal differentiation. Loss of FUBP1 in neural stem/progenitor cells (NPC) increased the number of cells fail to terminally differentiate. Knockdown of FUBP1 further blocks differentiation and promotes tumorigenesis collaboratively with the expression of neomorphic isocitrate dehydrorgenase 1 mutant IDH1R132H. Mechanistically FUBP1 is necessary for mini exon 8a splicing of neurospecific isoform of histone demethylase LSD1 (nLSD1). It binds to intron 8 of LSD1 together with a neuron-specific splicing complex containing NOVA1 and 2 and works synergistically with SRRM4 for the inclusion of mini exon 8a. The expression level of nLSD1 is decreased upon loss of FUBP1 in neural progenitors, impairing the terminal neuronal differentiation and maturation. Reinforcing nLSD1 expression in FUBP1-downregulated NPC restores terminal differentiation, establishing nLSD1 as an obligatory effector of FUBP1-dependent neuronal differentiation. Together, these findings establish a direct role for FUBP1 in neuronal differentiation and also uncover the mechanism for its tumor suppressor function in the nervous system. Citation Format: Inah Hwang, Dongqing Cao, Do-Yeon Kim, Tuo Zhang, Jian Hu, Yu Yao, Jihye Paik. Loss of FUBP1 impairs terminal neuronal differentiation and predisposes neural progenitors for transformation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2481.

Adenosine in the normal kidney significantly elevates in response to cellular damage. The renal A 3 adenosine receptor (A 3 AR) is up-regulated under stress, but the therapeutic effects of A 3 AR antagonists on chronic kidney disease are not fully understood. The present study examined the effect of LJ-1888 [(2 R ,3 R ,4 S )-2-[2-chloro-6-(3-iodobenzylamino)-9 H -purine-9-yl]-tetrahydrothiophene-3,4-diol], a newly developed potent, selective, species-independent, and orally active A 3 AR antagonist, on unilateral ureteral obstruction (UUO)–induced renal fibrosis. Pretreatment with LJ-1888 inhibited UUO-induced fibronectin and collagen I up-regulation in a dose-dependent manner. Masson's trichrome staining confirmed that LJ-1888 treatment effectively reduced UUO-induced interstitial collagen accumulation. Furthermore, delayed administration of LJ-1888 showed an equivalent therapeutic effect on tubulointerstitial fibrosis to that of losartan. Small-interfering A 3 AR transfection effectively inhibited transforming growth factor-β1 (TGF-β1)–induced fibronectin and collagen I up-regulation in proximal tubular cells similar to LJ-1888, confirming that the renoprotective effect of LJ-1888 resulted from A 3 AR blockade. UUO- or TGF-β1–induced c-Jun N-terminal kinase and extracellular signal–regulated kinase phosphorylation decreased significantly after LJ-1888 administration. A 3 AR blockade reduced UUO- or TGF-β1–induced up-regulation of lysyl oxidase, which induces cross-linking of extracellular matrix, suggesting that LJ-1888 may also regulate extracellular matrix accumulation via post-translational regulation. In conclusion, the present data demonstrate that the A 3 AR antagonist, LJ-1888, blocked the development and attenuated the progression of renal fibrosis, and they suggest that LJ-1888 may become a new therapeutic modality for renal interstitial fibrosis.

Aims The present study examined renoprotective effect of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSC) in diabetes. NRK-52E cells were utilized to determine the paracrine effect of hUCB-MSC. Methods hUCB was harvested with the mother's consent. MSC obtained from the hUCB were injected through the tail vein. Growth arrested and synchronized NRK-52E cells were stimulated with transforming growth factor-β1 (TGF-β1) in the presence of hUCB-MSC conditioned media. Results At 4 weeks after the streptozotocin (STZ) injection, diabetic rats showed significantly increased urinary protein excretion, renal and glomerular hypertrophy, fractional mesangial area, renal expression of TGF-β1 and α-smooth muscle actin, and collagen accumulation but decreased renal E-cadherin and bone morphogenic protein-7 expression, confirming diabetic renal injury. hUCB-MSC effectively prevented diabetic renal injury except renal and glomerular hypertrophy without a significant effect on blood glucose. CM-DiI-labeled hUCB-MSC and immunostaining of PKcs, a human nuclei antigen, confirmed a few engraftment of hUCB-MSC in diabetic kidneys. hUCB-MSC conditioned media inhibited TGF-β1-induced extracellular matrix upregulation and epithelial-to-mesenchymal transition in NRK-52E cells in a concentration-dependent manner. Conclusions These results demonstrate the renoprotective effect of hUCB-MSC in STZ-induced diabetic rats possibly through secretion of humoral factors and suggest hUCB-MSC as a possible treatment modality for diabetic renal injury.

Fibrosis of epithelial parenchymal organs and end-stage organ failure, the final common pathway of many progressive chronic diseases including chronic kidney disease, continue to increase worldwide and are a major determinant of morbidity and mortality. Fibrosis is an active biosynthetic healing response initiated to protect the tissue from injury through the timed release of proteins but leads to serious tissue damage when it becomes independent from the initiating stimulus. Massive deposition of extracellular matrix by accumulation of myofibroblasts and disruption of the normal tissue architecture are characteristic of tissue fibrosis. The highly conserved Wnt/beta-catenin signaling pathway is essential to embryonic development in general and kidney morphogenesis in particular by regulating the expression of target genes, most often through the transcription factor T cell factor (TCF) and/or lymphoid enhancer factor (LEF). Emerging evidence from studies of renal fibrosis suggests that altered Wnt/beta-catenin signaling is linked to the pathogenesis of renal fibrosis. The renoprotective properties of some currently available drugs might be attributable in part to inhibition of Wnt signaling. The development of orally active Wnt modulators will provide a potentially important pharmacological tool for further investigating the role of Wnt/beta-catenin signaling and might offer a novel therapeutic strategy in renal fibrosis.